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. 2002 Dec 1;368(Pt 2):535–543. doi: 10.1042/BJ20020226

Protein kinase C- and calcium-regulated pathways independently synergize with Gi pathways in agonist-induced fibrinogen receptor activation.

Todd M Quinton 1, Soochong Kim 1, Carol Dangelmaier 1, Robert T Dorsam 1, Jianguo Jin 1, James L Daniel 1, Satya P Kunapuli 1
PMCID: PMC1223015  PMID: 12215172

Abstract

Platelet fibrinogen receptor activation is a critical step in platelet plug formation. The fibrinogen receptor (integrin alphaIIbbeta3) is activated by agonist-mediated G(q) stimulation and resultant phospholipase C activation. We investigated the role of downstream signalling events from phospholipase C, namely the activation of protein kinase C (PKC) and rise in intracellular calcium, in agonist-induced fibrinogen receptor activation using Ro 31-8220 (a PKC inhibitor) or dimethyl BAPTA [5,5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N', N'-tetra-acetic acid], a high-affinity calcium chelator. All the experiments were performed with human platelets treated with aspirin, to avoid positive feedback from thromboxane A2. In the presence of Ro 31-8220, platelet aggregation caused by U46619 was completely inhibited while no effect or partial inhibition was seen with ADP and the thrombin-receptor-activating peptide SFLLRN, respectively. In the presence of intracellular dimethyl BAPTA, ADP- and U46619-induced aggregation and anti-alphaIIbbeta3 antibody PAC-1 binding were completely abolished. However, similar to the effects of Ro 31-8220, dimethyl BAPTA only partially inhibited SFLLRN-induced aggregation, and was accompanied by diminished dense-granule secretion. When either PKC activation or intracellular calcium release was abrogated, aggregation and fibrinogen receptor activation with U46619 or SFLLRN was partially restored by additional selective activation of the G(i) signalling pathway. In contrast, when both PKC activity and intracellular calcium increase were simultaneously inhibited, the complete inhibition of aggregation that occurred in response to either U46619 or SFLLRN could not be restored with concomitant G(i) signalling. We conclude that, while the PKC- and calcium-regulated signalling pathways are capable of inducing activating fibrinogen receptor independently and that each can synergize with G(i) signalling to cause irreversible fibrinogen receptor activation, both pathways act synergistically to effect irreversible fibrinogen receptor activation.

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Selected References

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  1. Atkinson B. T., Stafford M. J., Pears C. J., Watson S. P. Signalling events underlying platelet aggregation induced by the glycoprotein VI agonist convulxin. Eur J Biochem. 2001 Oct;268(20):5242–5248. doi: 10.1046/j.0014-2956.2001.02448.x. [DOI] [PubMed] [Google Scholar]
  2. Baldassare J. J., Henderson P. A., Burns D., Loomis C., Fisher G. J. Translocation of protein kinase C isozymes in thrombin-stimulated human platelets. Correlation with 1,2-diacylglycerol levels. J Biol Chem. 1992 Aug 5;267(22):15585–15590. [PubMed] [Google Scholar]
  3. Bauer M., Retzer M., Wilde J. I., Maschberger P., Essler M., Aepfelbacher M., Watson S. P., Siess W. Dichotomous regulation of myosin phosphorylation and shape change by Rho-kinase and calcium in intact human platelets. Blood. 1999 Sep 1;94(5):1665–1672. [PubMed] [Google Scholar]
  4. Benka M. L., Lee M., Wang G. R., Buckman S., Burlacu A., Cole L., DePina A., Dias P., Granger A., Grant B. The thrombin receptor in human platelets is coupled to a GTP binding protein of the G alpha q family. FEBS Lett. 1995 Apr 17;363(1-2):49–52. doi: 10.1016/0014-5793(95)00278-h. [DOI] [PubMed] [Google Scholar]
  5. Brass L. F., Manning D. R., Cichowski K., Abrams C. S. Signaling through G proteins in platelets: to the integrins and beyond. Thromb Haemost. 1997 Jul;78(1):581–589. [PubMed] [Google Scholar]
  6. Coughlin S. R. How the protease thrombin talks to cells. Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11023–11027. doi: 10.1073/pnas.96.20.11023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Crabos M., Imber R., Woodtli T., Fabbro D., Erne P. Different translocation of three distinct PKC isoforms with tumor-promoting phorbol ester in human platelets. Biochem Biophys Res Commun. 1991 Aug 15;178(3):878–883. doi: 10.1016/0006-291x(91)90973-b. [DOI] [PubMed] [Google Scholar]
  8. Daniel J. L., Dangelmaier C., Jin J., Ashby B., Smith J. B., Kunapuli S. P. Molecular basis for ADP-induced platelet activation. I. Evidence for three distinct ADP receptors on human platelets. J Biol Chem. 1998 Jan 23;273(4):2024–2029. doi: 10.1074/jbc.273.4.2024. [DOI] [PubMed] [Google Scholar]
  9. Daniel J. L., Dangelmaier C., Smith J. B. Evidence for a role for tyrosine phosphorylation of phospholipase C gamma 2 in collagen-induced platelet cytosolic calcium mobilization. Biochem J. 1994 Sep 1;302(Pt 2):617–622. doi: 10.1042/bj3020617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Djellas Y., Manganello J. M., Antonakis K., Le Breton G. C. Identification of Galpha13 as one of the G-proteins that couple to human platelet thromboxane A2 receptors. J Biol Chem. 1999 May 14;274(20):14325–14330. doi: 10.1074/jbc.274.20.14325. [DOI] [PubMed] [Google Scholar]
  11. Foster C. J., Prosser D. M., Agans J. M., Zhai Y., Smith M. D., Lachowicz J. E., Zhang F. L., Gustafson E., Monsma F. J., Jr, Wiekowski M. T. Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs. J Clin Invest. 2001 Jun;107(12):1591–1598. doi: 10.1172/JCI12242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gabbeta J., Yang X., Kowalska M. A., Sun L., Dhanasekaran N., Rao A. K. Platelet signal transduction defect with Galpha subunit dysfunction and diminished Galphaq in a patient with abnormal platelet responses. Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8750–8755. doi: 10.1073/pnas.94.16.8750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gabbeta J., Yang X., Sun L., McLane M. A., Niewiarowski S., Rao A. K. Abnormal inside-out signal transduction-dependent activation of glycoprotein IIb-IIIa in a patient with impaired pleckstrin phosphorylation. Blood. 1996 Feb 15;87(4):1368–1376. [PubMed] [Google Scholar]
  14. Grabarek J., Raychowdhury M., Ravid K., Kent K. C., Newman P. J., Ware J. A. Identification and functional characterization of protein kinase C isozymes in platelets and HEL cells. J Biol Chem. 1992 May 15;267(14):10011–10017. [PubMed] [Google Scholar]
  15. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  16. Hashimoto Y., Togo M., Tsukamoto K., Horie Y., Watanabe T., Kurokawa K. Protein kinase C-dependent and -independent mechanisms of dense granule exocytosis by human platelets. Biochim Biophys Acta. 1994 May 26;1222(1):56–62. doi: 10.1016/0167-4889(94)90024-8. [DOI] [PubMed] [Google Scholar]
  17. Hers I., Donath J., van Willigen G., Akkerman J. W. Differential involvement of tyrosine and serine/threonine kinases in platelet integrin alphaIIbbeta3 exposure. Arterioscler Thromb Vasc Biol. 1998 Mar;18(3):404–414. doi: 10.1161/01.atv.18.3.404. [DOI] [PubMed] [Google Scholar]
  18. Hollopeter G., Jantzen H. M., Vincent D., Li G., England L., Ramakrishnan V., Yang R. B., Nurden P., Nurden A., Julius D. Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature. 2001 Jan 11;409(6817):202–207. doi: 10.1038/35051599. [DOI] [PubMed] [Google Scholar]
  19. Hourani S. M., Cusack N. J. Pharmacological receptors on blood platelets. Pharmacol Rev. 1991 Sep;43(3):243–298. [PubMed] [Google Scholar]
  20. Jin J., Daniel J. L., Kunapuli S. P. Molecular basis for ADP-induced platelet activation. II. The P2Y1 receptor mediates ADP-induced intracellular calcium mobilization and shape change in platelets. J Biol Chem. 1998 Jan 23;273(4):2030–2034. doi: 10.1074/jbc.273.4.2030. [DOI] [PubMed] [Google Scholar]
  21. Jin J., Kunapuli S. P. Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci U S A. 1998 Jul 7;95(14):8070–8074. doi: 10.1073/pnas.95.14.8070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jin Jianguo, Quinton Todd M., Zhang Jin, Rittenhouse Susan E., Kunapuli Satya P. Adenosine diphosphate (ADP)-induced thromboxane A(2) generation in human platelets requires coordinated signaling through integrin alpha(IIb)beta(3) and ADP receptors. Blood. 2002 Jan 1;99(1):193–198. doi: 10.1182/blood.v99.1.193. [DOI] [PubMed] [Google Scholar]
  23. Lee S. B., Rao A. K., Lee K. H., Yang X., Bae Y. S., Rhee S. G. Decreased expression of phospholipase C-beta 2 isozyme in human platelets with impaired function. Blood. 1996 Sep 1;88(5):1684–1691. [PubMed] [Google Scholar]
  24. Mellor H., Parker P. J. The extended protein kinase C superfamily. Biochem J. 1998 Jun 1;332(Pt 2):281–292. doi: 10.1042/bj3320281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Offermanns S., Toombs C. F., Hu Y. H., Simon M. I. Defective platelet activation in G alpha(q)-deficient mice. Nature. 1997 Sep 11;389(6647):183–186. doi: 10.1038/38284. [DOI] [PubMed] [Google Scholar]
  26. Packham M. A. Role of platelets in thrombosis and hemostasis. Can J Physiol Pharmacol. 1994 Mar;72(3):278–284. doi: 10.1139/y94-043. [DOI] [PubMed] [Google Scholar]
  27. Paul B. Z., Daniel J. L., Kunapuli S. P. Platelet shape change is mediated by both calcium-dependent and -independent signaling pathways. Role of p160 Rho-associated coiled-coil-containing protein kinase in platelet shape change. J Biol Chem. 1999 Oct 1;274(40):28293–28300. doi: 10.1074/jbc.274.40.28293. [DOI] [PubMed] [Google Scholar]
  28. Paul B. Z., Jin J., Kunapuli S. P. Molecular mechanism of thromboxane A(2)-induced platelet aggregation. Essential role for p2t(ac) and alpha(2a) receptors. J Biol Chem. 1999 Oct 8;274(41):29108–29114. doi: 10.1074/jbc.274.41.29108. [DOI] [PubMed] [Google Scholar]
  29. Pulcinelli F. M., Ashby B., Gazzaniga P. P., Daniel J. L. Protein kinase C activation is not a key step in ADP-mediated exposure of fibrinogen receptors on human platelets. FEBS Lett. 1995 May 1;364(1):87–90. doi: 10.1016/0014-5793(95)00352-a. [DOI] [PubMed] [Google Scholar]
  30. Pulcinelli F. M., Ciampa M. T., Favilla M., Pignatelli P., Riondino S., Gazzaniga P. P. Concomitant activation of Gi protein-coupled receptor and protein kinase C or phospholipase C is required for platelet aggregation. FEBS Lett. 1999 Oct 22;460(1):37–40. doi: 10.1016/s0014-5793(99)01313-7. [DOI] [PubMed] [Google Scholar]
  31. Pulcinelli F. M., Pesciotti M., Pignatelli P., Riondino S., Gazzaniga P. P. Concomitant activation of Gi and Gq protein-coupled receptors does not require an increase in cytosolic calcium for platelet aggregation. FEBS Lett. 1998 Sep 11;435(1):115–118. doi: 10.1016/s0014-5793(98)01049-7. [DOI] [PubMed] [Google Scholar]
  32. Quinton Todd M., Ozdener Fatih, Dangelmaier Carol, Daniel James L., Kunapuli Satya P. Glycoprotein VI-mediated platelet fibrinogen receptor activation occurs through calcium-sensitive and PKC-sensitive pathways without a requirement for secreted ADP. Blood. 2002 May 1;99(9):3228–3234. doi: 10.1182/blood.v99.9.3228. [DOI] [PubMed] [Google Scholar]
  33. Rao A. K., Koike K., Willis J., Daniel J. L., Beckett C., Hassel B., Day H. J., Smith J. B., Holmsen H. Platelet secretion defect associated with impaired liberation of arachidonic acid and normal myosin light chain phosphorylation. Blood. 1984 Oct;64(4):914–921. [PubMed] [Google Scholar]
  34. Rao A. K., Kowalska M. A., Disa J. Impaired cytoplasmic ionized calcium mobilization in inherited platelet secretion defects. Blood. 1989 Aug 1;74(2):664–672. [PubMed] [Google Scholar]
  35. Saitoh M., Salzman E. W., Smith M., Ware J. A. Activation of protein kinase C in platelets by epinephrine and A23187: correlation with fibrinogen binding. Blood. 1989 Nov 1;74(6):2001–2006. [PubMed] [Google Scholar]
  36. Shattil S. J., Cunningham M., Wiedmer T., Zhao J., Sims P. J., Brass L. F. Regulation of glycoprotein IIb-IIIa receptor function studied with platelets permeabilized by the pore-forming complement proteins C5b-9. J Biol Chem. 1992 Sep 15;267(26):18424–18431. [PubMed] [Google Scholar]
  37. Shattil S. J., Kashiwagi H., Pampori N. Integrin signaling: the platelet paradigm. Blood. 1998 Apr 15;91(8):2645–2657. [PubMed] [Google Scholar]
  38. Trybulec M., Kowalska M. A., McLane M. A., Silver L., Lu W., Niewiarowski S. Exposure of platelet fibrinogen receptors by zinc ions: role of protein kinase C. Proc Soc Exp Biol Med. 1993 May;203(1):108–116. doi: 10.3181/00379727-203-43580. [DOI] [PubMed] [Google Scholar]
  39. Wang F., Naik U. P., Ehrlich Y. H., Freyberg Z., Osada S., Ohno S., Kuroki T., Suzuki K., Kornecki E. A new protein kinase C, nPKC eta', and nPKC theta are expressed in human platelets: involvement of nPKC eta' and nPKC theta in signal transduction stimulated by PAF. Biochem Biophys Res Commun. 1993 Feb 26;191(1):240–246. doi: 10.1006/bbrc.1993.1208. [DOI] [PubMed] [Google Scholar]
  40. Watson S. P., McNally J., Shipman L. J., Godfrey P. P. The action of the protein kinase C inhibitor, staurosporine, on human platelets. Evidence against a regulatory role for protein kinase C in the formation of inositol trisphosphate by thrombin. Biochem J. 1988 Jan 15;249(2):345–350. doi: 10.1042/bj2490345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wilkinson S. E., Parker P. J., Nixon J. S. Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem J. 1993 Sep 1;294(Pt 2):335–337. doi: 10.1042/bj2940335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yang X., Sun L., Ghosh S., Rao A. K. Human platelet signaling defect characterized by impaired production of inositol-1,4,5-triphosphate and phosphatidic acid and diminished Pleckstrin phosphorylation: evidence for defective phospholipase C activation. Blood. 1996 Sep 1;88(5):1676–1683. [PubMed] [Google Scholar]
  43. van Willigen G., Akkerman J. W. Protein kinase C and cyclic AMP regulate reversible exposure of binding sites for fibrinogen on the glycoprotein IIB-IIIA complex of human platelets. Biochem J. 1991 Jan 1;273(Pt 1):115–120. doi: 10.1042/bj2730115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. van Willigen G., Akkerman J. W. Regulation of glycoprotein IIB/IIIA exposure on platelets stimulated with alpha-thrombin. Blood. 1992 Jan 1;79(1):82–90. [PubMed] [Google Scholar]

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